The present invention relates generally to molecules such as peptides, polypeptides and proteins which carry epitopes and in particular B cell epitopes from antigenic proteins encoded by Hepatitis E Virus. These molecules are preferentially immunoreactive to convalescent or acute phase circulating antibodies to the Hepatitis E Virus and are useful in the development of diagnostic, therapeutic and prophylactic agents for Hepatitis E Virus.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Viral hepatitis results from infection with one of at least five very different viral agents. Available serological tests allow the diagnosis of acute hepatitis due to infection with Hepatitis A Virus (HAV) and Hepatitis B Virus (HBV). HBV is required for propagation of the delta agent, or Hepatitis D Virus (HDV); this co-infection results in a high proportion of cases progressing to chronic active hepatitis. The clinical and diagnostic exclusion of HAV and HBV has led to the recognition of other hepatitides that were formerly grouped together as non-A, non-B hepatitis [(NANBH)] (Prince et al., 1974; Feinstone et al., 1975; Tabor, 1985). NANBH is caused by more than one viral agent and can be transmitted by either parenteral or fecal/oral routes (Bradley, 1990a; Reyes and Baroudy, 1991).
The cloning of a blood-borne agent, termed Hepatitis C Virus (HCV), led to the development of a specific assay for circulating antibody to HCV (Choo et al., 1989; Kuo et al., 1989; Kubo et al., 1989, Maeno et al., 1990). This assay predominantly detects infections at the chronic stage, but has facilitated the identification of HCV as the cause of up to 90% of parenterally transmitted NANBH. A second epidemiologically distinct form of NANBH has been shown to occur in both epidemic and sporadic patterns in developing countries and is referred to as enterically transmitted non-A, non-B hepatitis (ET-NANBH) due to its water-borne mode of virus transmission and presumed enteric route of infection (Khuroo, 1980; Wong et al., 1980). ET-NANBH has been documented in India, Pakistan, Burma, USSR, Costa Rica, Mexico and countries in Africa where epidemic outbreaks can generally be traced to fecal contamination of drinking water (Bradley and Maynard, 1986; Bradley, 1990b). The causative viral agent was previously shown to passage successfully in cynomolgus macaques (cyno) and tamarins with typical liver enzyme elevations and recovery of morphologically similar 27 to 34 mm virus like particles from the feces of clinical specimens and experimental animals (Balayan et al., 1983; Andiaparidze et al., 1986; Bradley et al., 1987; Arankalle et al., 1988).
Reyes et al. (1990) recently reported the isolation of a partial cDNA clone from the virus responsible for ET-NANBH, and termed the newly identified agent the Hepatitis E Virus (HEV). The expressions xe2x80x9centerically transmitted non-A, non-B hepatitisxe2x80x9d, xe2x80x9cET-NANBHxe2x80x9d, xe2x80x9cHepatitis E Virusxe2x80x9d and xe2x80x9cHEVxe2x80x9d are used interchangeably herein to refer to a virus or type of virus which is capable of causing infectious hepatitis from contaminated water, which is transmissible and capable of passage in cynomologus macaques and tramarins, which is serologically distinct from HAV, HBV, HCV and HDV and which comprises a genomic nucleotide sequence, a portion of which, at least is homologous to or substantially similar to or capable of hybridizing under low stringency conditions to the all or part of the nucleotide sequences set forth in FIG. 1 and/or FIG. 2. Preferred homologies and a definition of stringency conditions are set forth below.
The HEV clone was from a Burma isolate of the virus and hybridised with cDNA made from five other distinct geographic isolates. These molecular epidemiological findings are consistent with the available serologic data based on the use of immune electron microscopy and imunofluorescence blocking studies that indicate a single major agent is responsible for the majority of ET-NANBH seen worldwide (Purcell and Ticehurst, 1988; Bradley et al. 1988a; Krawczynski and Bradley, 1989). Tam et al. (1991) subsequently reported on the molecular cloning of the complete HEV (Burma; B) viral clone together with the deduced amino acid sequences.
HEV has a single stranded genome of polyadenylated RNA of positive polarity which encodes three open reading frames (ORF""s) designated ORF1, ORF2 and ORF3. Open reading frame 2 and ORF3 are partially overlapping in different reading frames and are thought to encode structural proteins of the virus. Open reading frame 1, on the other hand, encodes replicative proteins and overlaps ORF3 by one nucleotide.
Despite the availability of immunoassays for the detection of antibodies to some strains of HEV, it has been observed that IgM and IgG antibody titres wane rapidly following infection. Consequently, it has proven difficult to interpret serological surveys from both endemic and non-endemic areas. Furthermore, the relationship between antibody interactivity and immunity to reinfection remains unclear. These and other factors have delayed the development of suitable diagnostic and therapeutic protocols for the HEV, for which a need clearly exists. In particular, it would be most beneficial to develop an assay which could distinguish between acute phase antibodies (contemporary antibodies) generated in response to HEV infection and convalescent phase antibodies, which remain in the circulatory and/or secretory system after infection and may contribute to immunity to reinfection.
In work leading up to the present invention, the inventors sought to identify epitopes, and in particular B cell epitopes, on peptides, polypeptides and proteins encoded within the HEV genome in order to improve upon current diagnostic procedures and to further develop therapeutic and prophylactic compositions for HEV. In accordance with the present invention, peptides, polypeptides and proteins, were recombinantly expressed from nucleic acid molecules derived from ORFs in the HEV genome. The epitopic and in particular B cell epitopic regions within these molecules were identified on the basis of interactivity to antibodies specific to HEV, thereby forming the basis for a new range of diagnostic, therapeutic and prophylactic procedures for HEV. Interestingly, the inventors have determined that one portion of a molecule encoded by an ORF may inhibit or otherwise reduce the immunointeractivity of another portion of the same molecule. They have further determined that the inhibitory effect may be overcome by using non-full length molecules or reducing the inhibitory effect by physical or chemical processes.
Accordingly, one aspect of the present invention provides a recombinant molecule encoded by a sequence of nucleotides selected from the list consisting of open reading frame (ORF) 2 and ORF 3 of Hepatitis E Virus (HEV) or a mutant or derivative of said ORF2 or ORF3. More particularly, the present invention provides a recombinant molecule encoded by a sequence of nucleotides comprising xe2x80x9cORF3xe2x80x9d or a part of xe2x80x9cORF2xe2x80x9d and which molecule is preferentially immunologically interactive in either convalescent phase or acute phase antibodies to HEV are conveniently described respectively as those regions of the HEV genome beginning at nucleotide 5106 extending 369 bases and terminating at nucleotide 5474 (ORF3) and that region beginning at nucleotide 5147 extending 1980 bases and terminating 68 bases upstream of the poly(A) tail (ORF2) using the numbering system of Tam et al. (1991).
The term xe2x80x9crecombinant moleculexe2x80x9d in this context includes a peptide, polypeptide, protein or a chemical equivalent thereof. The recombinant molecule may or may not be glycosylated. A recombinant molecule also extends to a fusion between an HEV derived peptide, polypeptide or protein and a peptide, polypeptide or protein of non-HEV origin such as but not limited to glutathione-S-transferase (GST) or its derivatives, polylysine, polyhistidine, thioredoxin or any other molecule capable of bioaffinity chromatography.
In accordance with the present invention, it has been surprisingly discovered that recombinant molecules encoded by ORF2 and in particular the 3xe2x80x2 end of ORF2 and ORF3 are capable of interacting with convalescent phase antibodies to HEV whereas recombinant molecules encoded by the 5xe2x80x2 end of ORF2 are interactive more preferentially with acute phase antibodies generated to the virus particle per se. According to this aspect of the present invention, there is provided a recombinant peptide or polypeptide which is preferentially immunologically reactive with convalescent phase antibodies generated during HEV infection. In a related aspect of the present invention, there is provided a recombinant peptide or polypeptide which is preferentially immunologically interactive with acute phase antibodies generated during HEV infection.
As used herein, the 3xe2x80x2 end of an ORF (e.g. ORF2) is considered the carboxy (C)-terminal end portion of the molecule encoded by the ORF and the 5xe2x80x2 end is considered the amino (N)-terminal end portion.
More particularly, it has further been surprisingly discovered that the 5xe2x80x2 (i.e. the C-terminal) end portion of ORF2 exhibits greater immunointeractivity to acute phase antibodies to HEV and the 5xe2x80x2 (i.e. N-terminal) end portion of ORF2 exhibits greater immunointeractivity to convalescent phase antibodies to HEV, when present on the same molecule, however, the N-terminal end portion appears to inhibit or reduce the immunointeractivity of the C-terminal end portion.
Accordingly, another aspect of the present invention contemplates a recombinant molecule encoded by a sequence of nucleotides comprising ORF3 or a non-full length ORF2 derived from the 3xe2x80x2 end of ORF2 of HEV and wherein said recombinant molecule is preferentially interactive with convalescent phase antibodies to HEV. In a related aspect there is provided a recombinant molecule encoded by a sequence of nucleotides comprising a non-length ORF2 derived from the 5xe2x80x2 end of ORF2 of HEV and wherein said recombinant molecule is preferentially interactive with acute phase antibodies to HEV.
In one embodiment the sequence of nucleotides is from the 5xe2x80x2 end of ORF2 and encodes a molecule more interactive with acute phase antibodies relative to convalescent phase antibodies. In another embodiment, the nucleotide sequence is from ORF3 or the 3xe2x80x2 end of ORF2 and encodes a molecule more interactive with convalescent phase antibodies relative to acute phase antibodies. The preferred nucleotide sequence of the 5xe2x80x2 end of ORF2 includes the clones 2.3 and 2.4 defined in FIG. 5.
The recombinant molecule may also be encoded by a nucleotide sequence comprising the full length ORF2 but modified to permit interactivity to both convalescent and acute phase antibodies to HEV. Such modification includes chemical and physical treatments to expose epitopes, for example by denaturing followed by renaturing of the molecule or the use of antagonists to prevent the inhibition of, for example, the N-terminal end portion over the C-terminal end portion binding to HEV antibodies.
xe2x80x9cAcute and xe2x80x9cconvalescentxe2x80x9d phases are used in their broadest sense to mean respectively the period during infection (acute) and the period beginning from after the peak of infection extending through a convalescent period to a period post infection. The acute phase of infection can conveniently be considered, for example, to be approximately 3 to 6 months following exposure to HEV and the convalescent phase is generally after this period.
Yet another aspect of the present invention is directed to a recombinant polypeptide comprising first and second amino acid sequences wherein said first amino acid sequence is encoded by a nucleotide sequence selected from or within ORF2 and ORF 3 of HEV and wherein said second amino acid sequence is a non-HEV encoded peptide, polypeptide or protein such as but not exclusively GST. Preferably, the first amino acid sequence is encoded by ORF3 or the 3xe2x80x2 end of ORF2 and is interactive with convalescent phase antibodies to HEV. Alternatively, the first amino acid sequence is from the 5xe2x80x2 end portion of ORF2 and is preferably interactive with acute phase antibodies to HEV.
The presence of selectively or preferentially immunologically reactive antigens provides a means of distinguishing between past (convalescent) and acute (contemporary) phase infection with HEV. Accordingly, the present invention also extends to a method for distinguishing between convalescent and acute phase infection with HEV in an individual which method measures the reactivity of serum to immune interactive recombinant molecule encoded by ORF2 and/or ORF3 or more preferably parts thereof. Most preferably, these parts include the 3xe2x80x2 end portion of ORF2 or the 5xe2x80x2 end portion of ORF2 or is a chemically modified form of a fill length molecule such as to expose epitopes on both the N- and C-termini.
The present invention also provides an isolated nucleic acid molecule such as DNA or eDNA molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a peptide, polypeptide or protein interactive with acute phase antibodies to HEV. In an alternative embodiment, the peptide, polypeptide or protein is interactive with both convalescent and/or acute phase circulating antibodies to HEV. Accordingly, this aspect of the present invention provides a recombinant molecule carrying a B cell epitope of a polypeptide or protein encoded by ORF2 or ORF3 or more preferably a part thereof. More particularly, the sequence of nucleotides is selected from a sequence comprising or within nucleotides 5147 to 7129 (ORF2) and 5106 to 5474 (ORF3) in the HEV genome using the nucleotide numbering system of Tam et al (1991) or a nucleotide region substantially equivalent thereto. Preferably, the nucleotide sequence is operably linked to an expression control sequence such as in the form of an expression vector.
The recombinant molecule according to this aspect of the present invention is generally an isolated nucleic acid molecule comprising DNA (eg cDNA or genonic DNA) or mRNA. The recombinant molecule may be isolated directly from the HEV genome or may be generated in vitro by, for example, the stepwise addition of nucleotides or groups of nucleotides. The present invention further extends to a host cell such as bacterium, yeast, mammalian or insect cell transformed with such a recombinant molecule. A preferred mammalian cell is the Chinese Hamster Ovary (CHO) cell line. A preferred bacterium is E. coli. Preferably, the nucleic acid molecules are DNA, at least parts of which have a nucleotide sequence substantially corresponding to all or part of the nucleotide sequence shown in FIGS. 1 (SEQ ID NO: 1) or 2 (SEQ ID NO: 3) or a part, fragment, derivative, homologue or analogue thereof or one or more sequences complementary thereto. The present invention, however also extends to any single or multiple nucleotide substitutions, deletions and/or additions to the sequence shown in FIGS. 1 or 2 and which still encode an epitopic region of the HEV genome or fragment or derivative thereof having the requisite antigenic profile and interactive with antibodies to HEV. Most preferably, the nucleotide sequence encode either the 3xe2x80x2 end of ORF2 or the 5xe2x80x2 end of ORF2 such that the nucleotide sequence encodes a peptide or polypeptide which interacts preferably with convalescent phase antibodies or acute phase antibodies, respectively.
Furthermore, when the nucleic acid molecule is RNA, the ribonucleotide sequence will, in a preferred embodiment, be substantially complementary to one or more of the nucleotide sequences shown in FIGS. 1 or 2 a part, fragment, derivative, homologue or analogue thereof. Another aspect of this invention is directed to a synthetic (e.g. recombinant) peptide, polypeptide or protein which is immunologically interactive with convalescent phase antibodies generating during HEV infection. Furthermore, the invention is further directed to a synthetic (eg: recombinant) peptide, polypeptide or protein which is immunologically interactive with acute and convalescent circulating antibodies generated during HEV infection.
Such synthetic peptides, polypeptides or proteins may, for example, be prepared by recombinant means such as by the expression of a host cell transformed with the recombinant molecules described above. The peptide, polypeptide or protein may be fused to another peptide, polypeptide or protein. Alternatively, it may be prepared by chemical synthesis, such as by the Merrifield solid-phase synthesis procedure. Furthermore, although synthetic or fragments thereof represent a preferred embodiment, the present invention also extends to biologically pure preparations of the naturally occurring epitopes or their fragments. By xe2x80x9cbiologically purexe2x80x9d is meant a preparation of at least 60%, preferably at least 70%, more preferably at least 80% and still more preferably at least 90% by weight peptide, polypeptide or protein.
Another aspect of the present invention contemplates a peptide, polypeptide or protein carrying a B cell epitope interactive with antibodies to HEV, said peptide, polypeptide or protein being encoded by a sequence of nucleotides capable of hybridising under low stringency conditions to one or more regions of the nucleotide sequence set forth in FIG. 1 (SEQ ID NO: 1) or 2 (SEQ ID NO: 3).
More particularly, one aspect of this embodiment is directed to a nucleic acid molecule comprising a sequence of nucleotides which:
(i) encodes a peptide, polypeptide or protein interactive with acute and/or convalescent phase antibodies to HEV; and
(ii) is capable of hybridizing under low stringency conditions to all or part of the nucleotide sequence set forth in FIG. 1 (SEQ ID NO: 1) under low stringency conditions.
In a related aspect, the nucleotide sequence encodes a peptide, polypeptide or protein interactive with acute phase antibodies and is capable of hybridizing under low stringency conditions to all or part of the nucleotide sequence set forth in FIG. 1 or FIG. 2.
In a preferred embodiment, the nucleotide sequence is capable of hybridizing to the 5xe2x80x2 end of ORF2 and encodes a molecule capable of preferential interaction with acute phase antibodies to HEV. Most preferably, the nucleotide sequence is sequence 2.3 or 2.4 of FIG. 5.
Another aspect of this embodiment of the present invention provides a recombinant peptide, polypeptide or protein encoded by a nucleotide sequence capable of hybridizing under low stringency conditions to all or part of the nucleotide sequence set forth in FIG. 1 (SEQ ID NO: 1) or FIG. 2 (SEQ ID NO: 3). This aspect of the present invention includes recombinant or synthetic forms of naturally occurring proteins encoded by the aforementioned nucleotide sequences as well as peptide, polypeptide or protein derivatives thereof which are encoded by modified nucleotide sequences still capable of hybridizing to the nucleotide sequence in FIG. 1 (SEQ ID NO: 1) or 2 (SEQ ID NO: 3) and which derivatives are interactive with acute and/or convalescent phase antibodies to HEV.
For the purposes of defining the level of stringency, reference can conveniently be made to Maniatis et al (1982) at pages 387-389 which is herein incorporated by reference where the washing step disclosed is considered high stringency. A low stringency is defined herein as being in 4-6xc3x97SSC/0.1-0.5% w/v SDS at 37-45xc2x0 C. for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridization, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 1-4xc3x97SSC/0.25-0.5% w/v SDS at xe2x89xa745xc2x0 C. for 2-3 hours or high stringency conditions which are considered herein to be 0.1-1xc3x97SSC/0.1% w/v SDS at xe2x89xa760xc2x0 C. for 1-3 hours.
In yet a further aspect, the present invention contemplates a peptide, polypeptide or protein carrying a B cell epitope interactive with antibodies to HEV, said peptide, polypeptide or protein having a substantially similar amino acid sequence to one or more regions of the amino acid sequences set forth in FIG. 1 (SEQ ID NO: 2) or 2 (SEQ ID NO: 4). By xe2x80x9csubstantially similarxe2x80x9d is meant a sequence having at least 50%, preferably at least 60%, more preferably at least 75% and still more preferably at least 90% homology (i.e. similarity) with the sequences set forth in FIG. 1 (SEQ ID NO: 2) or 2 (SEQ ID NO: 4) at the amino acid level. Preferably, the peptide, polypeptide or protein comprise, the N-terminal encoding region of ORF2 which is preferentially interactive with acute phase antibodies to HEV. Alternatively, the peptide, polypeptide or protein comprises the C-terminal encoding region of ORF2 which is preferentially interactive with convalescent phase antibodies to HEV. In a further alternative, the peptide, polypeptide or protein is a full length ORF encoded molecule but chemically or physically modified or disrupted to expose both the N- and C-terminal epitopes.
The present invention also extends to a method for maintaining interactive conformation of synthetic (eg recombinant) peptides or polypeptides wherein the synthetic peptides or polypeptides are further purified after undergoing a denaturation procedure and subsequently renatured. By way of example the synthetic peptide may be denatured by adding denaturing concentrations of buffers including but not limited to, sodium dodecyl sulphate (SDS), 2-mercaptoethanol, or urea. Purification of denatured protein can be undertaken by procedures well known to the skilled artisan including but not limited to procedures for electrophoretic resolution on polyacrylamide gels or size exclusion chromatography. The denaturing buffers are removed by any number of means including but not limited to dilution, dialysis, chromatography or by binding to a solid support, for example membrane support or microtitre plate.
Accordingly, another aspect of the present invention contemplates a method of enhancing the immunointeractivity of a polypeptide or protein encoded by ORF 2 of HEV, said method comprising chemically or physically modifying the polypeptide or protein to enable both convalescent and acute phase antibodies to HEV to bind to said polypeptide or protein without substantial N-terminal inhibition of C-terminal binding to convalescent phase antibodies to HEV.
In a most preferred embodiment, the present invention extends to naturally occurring or synthetic (eg recombinant) peptide, polypeptide or protein corresponding to an approximately 30 KDa protein from the ORF2 region and/or an approximately 14 KDa protein from the ORF3 region and to nucleotide sequences coding for same as well as to fragments, derivatives, homologues or immunological relatives thereof. By xe2x80x9cderivativesxe2x80x9d is meant to include any single or multiple amino acid substitution, deletion and/or addition relative to the naturally occurring sequence or to the sequences as shown in FIGS. 1 (SEQ ID NO: 2) and 2 (SEQ ID NO: 4) and including any single or multiple substitution, deletion and/or addition to other molecules associated with the peptide or polypeptide including carbohydrate, lipid and/or other proteinacious moieties. Such derivatives, therefore, include glycosylated or non-glycosylated forms or molecules with altered glycosylation patterns.
The present invention is also directed to a method for distinguishing past (i.e. convalescent phase) or contemporary (i.e. acute phase) infection with HEV whether or not by natural infection or following vaccination, whereby the antisera from infected individuals are preferentially immunologically distinguished by interactive peptides of ORF3 or the N-terminus of ORF2. Alternatively, such antisera are preferably immunologically distinguished by interactive peptides of the C-terminal end portion of ORF2.
The present invention also contemplates a method for the detection of antibodies associated with HEV which method comprises contacting a peptide or polypeptide corresponding to all, or an antigenic portion of the epitopic regions of the HEV genome or a derivative thereof with a biological sample from a patient to be tested for a time and under conditions sufficient for a complex to form between the peptide or polypeptide and an antibody reactive to HEV and then detecting the complex. Preferably, the biological sample is serum, a blood derived component such as whole blood, serum or plasma or a secretory component such as saliva. Even more preferably, the peptide or polypeptide is immobilised onto a solid support before, during or after contact with the serum. Methods of detection are well known and include calorimetric, fluorometric and radioactive procedures. Other detection means can also be used such as involving immunoblotting. An example of such immunoblotting techniques includes the well known Western Blot technique. This assay can be varied in any number of ways without departing from the scope of the present invention. Another assay contemplated herein to be useful is the red cell agglutination assay as described by Wilson et al. (1991).
The present invention also extends to the use of peptides or polypeptide corresponding to the epitopic regions of the HEV genome or antigenic fragments thereof, as antigens in a diagnostic test for HEV, or for screening asymptomatic individuals by detection or determination of the titre of antibodies in a patient""s serum, for example using enzyme-linked immunoassay or radio immunoassay (RIA) technology or an agglutination assay using antigen-coated beads or the like.
This aspect of the present invention may conveniently be carried out by the detection and/or determination of the titre of antibodies in a biological sample (e.g. whole blood, plasma, serum or saliva) from a human subject, said method comprising contacting said sample with peptides or polypeptides corresponding to epitopic regions of the HEV genome or a fragment or derivative thereof for a time and under conditions sufficient for a complex to form between the peptide or polypeptide and an antibody reactive to HEV and then detecting the complex and/or amount of peptide or polypeptide which has been bound in the complex. Preferably, the peptide or polypeptide is immobilised onto a solid support before, during or after contact with the sample and the peptides or polypeptides are as hereinbefore defined.
Alternatively, HEV infection may be detected or at least a negative result re-confirmed by screening for HEV associated immune complexes. It is possible, for example, that a negative antibody result could have been caused by antibodies forming complexes with HEV thereby not being available for binding in the aforementioned assay. To conveniently detect HEV immune complexes, serum or other biological fluid is contacted with an anti-HEV antibody (e.g. a monoclonal antibody) for a time and under conditions sufficient for a HEV-antibody immune complex to bind. Preferably, the anti-HEV antibody is first immobilised onto a solid support. An anti-immunoglobulin antibody, generally with a label or other reporter molecule attached, is then used to screen for the antibody component of the HEV complex.
One skilled in the art will immediately recognise that the assays as contemplated herein may be modified without departing from the scope of the present invention. All such modifications and variations of these assays are encompassed by the present invention.
The presence of HEV convalescent and/or acute phase antibodies in a subject""s biological fluid (such as whole blood, plasma, serum or saliva) can be detected using a wide range of immunoassay techniques such as those described in U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. This includes both single-site and two-site, or xe2x80x9csandwichxe2x80x9d, assays of the non-competitive types, as well as in the traditional competitive binding assays. Sandwich assays are particularly useful and include ELISA dip-stick assays and Immunofluorometric assays (IFA). Competitive binding assays include radioimmunoassays (RIA). Western blot assays may also be used. The choice of assay will depend on the sensitivity required, materials available and the biological sample employed.
A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in one assay, an unlabelled peptide, polypeptide or protein (hereinafter abbreviated to xe2x80x9cpolypeptidexe2x80x9d) capable of preferably binding to acute or convalescent phase antibodies to HEV is immobilised onto a solid substrate and the biological sample to be tested for HEV antibodies brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of a polypeptide-antibody primary complex, an immunoglobulin specific antibody, labelled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing time sufficient for the formation of a secondary complex of polypeptide-antibody-labelled antibody. Any unreacted material is washed away, and the presence of the VPF is determined by observation of a signal produced by the reporter molecule on the second antibody. The results may either be qualitative, by simple observation of the visible signal or may be quantitated by comparing with a control sample containing known amounts of HEV antibody. Variations of this assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound polypeptide, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound polypeptide. These techniques are well known to those skilled in the art, and the possibility of variations will be readily apparent. The antibodies used above may be monoclonal or polyclonal.
In this assay, the HEV derived polypeptide or antigenic parts thereof is either covalently or passively bound to a solid surface. The solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microtitre plates, or any other surface suitable for conducting an immunoassay such as a dipstick. The binding processes are well-known in the art and generally consist of cross-linking, covalently binding or physically adsorbing the molecule to the insoluble carrier.
By xe2x80x9creporter moleculexe2x80x9d, as used in the present specification, is meant a molecule which, by its chemical nature, produces an analytically identifiable signal which allows the detection of an HEV polypeptide-antibody complex. The most commonly used reporter molecules in this type of assay include enzymes, fluorophores and radionuclide containing molecules (i.e. radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the antimmunoglobulin antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, xcex2-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change.
Alternatively, fluorescent compounds, such as fluorescein, anthanide such as europium and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in an enzyme immunoassay (EIA), the fluorescent labelled antibody is allowed to bind to the polypeptide in the polypeptide-antibody complex. After washing off the unbound reagent the remaining complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent molecules or bioluminescent molecules may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit the required purpose.
Accordingly, one aspect of the present invention contemplates a method of detecting convalescent and/or acute phase antibodies in HEV in a biological sample, said method comprising the steps of contacting said biological sample with an immobilised polypeptide preferably interactive with acute phase or convalescent phase antibodies to HEV for a time and under conditions sufficient for a polypeptide-antibody complex to form and subjecting said complex to a detecting means. The latter complex may be detected by, for example, the addition of an antimmunoglobulin antibody labelled with a reporter molecule.
Alternatively, a competitive immunoassay may be used. The most convenient assay of this type is a radioimmunoassay (RIA). In another assay, the red cell agglutination test may be employed (see Wilson et al. 1991).
The invention also extends to use of the peptides and/or polypeptides, or fragments, or derivatives of the present invention in the treatment or prophylaxis of patients.
As contemplated herein prophylaxis may be achieved in a patient by immunising a patient with a vaccine containing antigens corresponding to the epitopic regions of the HEV genome and a suitable adjuvant sufficient to elicit an antibody response in a patient. Methods for production of vaccines are well known to those skilled in the art and by way of example a convenient reference is Remington""s Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. Preferred HEV antigens include the N-terminal region of ORF2 or the C-terminal region of ORF2.
Due to the nature of the epitopic region(s) of the HEV antigen it should therefore be possible to discriminate between naturally infected individuals and vaccinated individuals.
The present invention encompasses other forms of kits and diagnostic assays including a kit comprising a container adapted to contain a synthetic peptide or polypeptide corresponding to the epitopic region of the HEV genome or its fragments, derivatives, homologues and/or immunological relatives. Preferred antigens are the N-terminal region of ORF2 and the C-terminal region of ORF2 of HEV. The kit may contain a second container adapted to contain or receive a sample to be tested. A third container may be present adapted to contain reagents for detecting HEV-antibody complexes. Alternatively, where the kit is to detect HEV immune complexes, the kit may comprise one or more containers (e.g. wells) adapted to contain a HEV specific antibody (e.g. a monoclonal antibody). Additional containers with the kit may then contain receptacles for receiving fluid samples and a labelled antibody.
In further accordance with the present invention, expression of the cDNA insert encoding the epitopic regions of HEV described herein or fragments thereof, may be achieved in a number of different ways.
As an example, successful expression of the antigens encoding the epitopic regions of HEV as a fusion protein can be achieved using the pGEX vectors which give expression of glutathione S-transferase fusion proteins, using E. coli as the host cells. Expression could also be achieved, by way of example, using pEV vectors or the polyhistidine expression vectors again using E. coli as the host cells. Any other suitable fusion protein capable of use in a bioaffinity chromatography may also be employed such as polylysine, FLAG and thioredoxin. Alternatively, the epitopic region of HEV may be expressed as a non-fused polypeptide, by using appropriate vector and host cell combinations. Other vector and host cell combinations which can be used in accordance with the present invention including a number of well described yeast shuttle vectors for use in yeast cells, or eukaryotic vectors useful in continuous cell lines, (eg CHO cells) yeast cells and/or insect cells or transgenic animals.
The present invention is applicable to any strain of HEV which has a genomic organisation comprising open reading frames equivalent to ORF2 and ORF3 as defined by Tam et al (1991). Of the strains isolated to date, any strain of HEV is useful in the practice of the present invention.
The present invention will now be further described with reference to the following non-limiting Figures and Examples.